Lipoproteins allow for transport of insoluble lipids in aqueous environments and transport the majority of cholesterol and triglyceride in the bloodstream. Alteration of normal lipoprotein levels can lead to human diseases, including atherosclerosis and coronary heart disease, one of the leading causes of mortality in developed countries. Dietary lipids are digested and taken to the endoplasmic reticulum (ER) in intestinal cells where they are processed before secretion to the bloodstream. An essential step of lipoprotein secretion is COPII-dependent transit from the ER to the Golgi in the form of chylomicrons in the intestine and very low density lipoproteins (VLDL) in the liver. These lipoprotein cargos are actually larger than canonical COPII vesicles, and thus pose a special problem for the secretory machinery. Despite the paramount importance of this process to human health and basic cell biology, the molecular mechanism of how COPII enables large lipoprotein cargo export from the ER is largely unknown. Chylomicron Retention Disease/Anderson's Disease (CMRD), a disease where newly formed lipoproteins are unable to exit from intestinal cells, offers an entry point to address this question. CMRD patients carry mutations in SAR1B, a component of the COPII coat; however no patients were identified with mutations in its highly conserved paralog, SAR1A. The functional difference between the two SAR1 paralogs may provide important insights into the mechanism of COPII secretion of large lipoprotein cargos. Because other COPII components have paralog-specific functions, these components, and their effectors, may also have essential roles in lipoprotein secretion. In order to elucidate the mechanisms by which large lipoprotein cargos are secreted, we will: 1) Define the amino acid residues responsible for functional differences between SAR1 paralogs and their effect on COPII coat formation and VLDL secretion. 2) Use in vitro transport vesicle budding assays to determine the requirements for specific COPII coat components for VLDL secretion. 3) Determine whether KLHL12, which ubiquitinates COPII component SEC31A and alters its ability to secrete the large cargo collagen, is required for transport of large lipoproteins. The experiments proposed here will provide novel information about the mechanisms of intracellular transport large lipoprotein cargos and the unique roles of COPII component paralogs. This information has implications for the etiology of CMRD and may provide insights into how to regulate VLDL secretion as a potential treatment for atherosclerosis and other human diseases.